Temporal relationship of autophagy and apoptosis in neurons challenged by low molecular weight β‐amyloid peptide

Alzheimer's disease (AD) is an aging‐related progressive neurodegenerative disorder. Previous studies suggested that various soluble Aβ species are neurotoxic and able to activate apoptosis and autophagy, the type I and type II programmed cell death, respectively. However, the sequential and functional relationships between these two cellular events remain elusive. Here we report that low molecular weight Aβ triggered cleavage of caspase 3 and poly (ADP‐ribose) polymerase to cause neuronal apoptosis in rat cortical neurons. On the other hand, Aβ activated autophagy by inducing autophagic vesicle formation and autophagy related gene 12 (ATG12), and up‐regulated the lysoso‐mal machinery for the degradation of autophagosomes. Moreover, we demonstrated that activation of autophagy by Aβ preceded that of apoptosis, with death associated protein kinase phosphorylation as the potential molecular link. More importantly, under Aβ toxicity, neurons exhibiting high level of autophagosome formation were absent of apoptotic features, and inhibition of autophagy by 3‐methylade‐nine advanced neuronal apoptosis, suggesting that autophagy can protect neurons from Aβ‐induced apoptosis.     

Cyclin D1与细胞周期调控

细胞周期是细胞生命活动中一个最重要的过程,其关键是G1 期的启动.细胞周期蛋白(Cyclin)、细胞周期蛋白依赖性激酶(CDKs)和CDK抑制因子(CKIs)是参与钿胞周期调控的主要因子.Cyclin D1是调控细胞周期G1期的关键蛋白,是一个比其他Cyclins更加敏感的指标,对细胞周期调控至关重要.综述Cyclin D1的结构和功能及其在肿瘤组织中的表达特征,初步分析Cyclin D在昆虫细胞周期调控的研究.     

PCR和分离培养同时检测儿童呼吸道感染的支原体

为了解广州市儿童呼吸道支原体感染情况,用一条共同的上游引物,二条特异性的下游引物建立的ＰＣＲ方法能同时扩增ＭＰ的６９１ｂｐ和ＭＧ的４３８ｂｐ粘附因子基因片段,但不会扩增其他支原体和细菌的ＤＮＡ。     

Evolution of the Hedgehog Gene Family

Effective intercellular communication is an important feature in the development of multicellular organisms. Secreted hedgehog (hh) protein is essential for both long- and short-range cellular signaling required for body pattern formation in animals. In a molecular evolutionary study, we find that the vertebrate homologs of the Drosophila hh gene arose by two gene duplications: the first gave rise to Desert hh, whereas the second produced the Indian and Sonic hh genes. Both duplications occurred before the emergence of vertebrates and probably before the evolution of chordates. The amino-terminal fragment of the hh precursor, crucial in long- and short-range intercellular communication, evolves two to four times slower than the carboxyl-terminal fragment in both Drosophila hh and its vertebrate homologues, suggesting conservation of mechanism of hh action in animals. A majority of amino acid substitutions in the amino- and carboxyl-terminal fragments are conservative, but the carboxyl-terminal domain has undergone extensive insertion-deletion events while maintaining its autocleavage protease activity. Our results point to similarity of evolutionary constraints among sites of Drosophila and vertebrate hh homologs and suggest some future directions for understanding the role of hh genes in the evolution of developmental complexity in animals.     

蛋白质及多肽的C端分析

本文采用（异）硫氰酸化学法,以乙酸酐为活化试剂,四丁铵硫氰酸为偶联试剂,溴甲基萘为烷化试剂,将蛋白质或多肽的Ｃ端依次转化并裂解为ＡＴＨ－氨基酸,在２５４ｎｍ进行检测。分析了若干种天然的、基因工程表达的蛋白质或多肽,测定了不同长度的Ｃ端序列,并确证了Ｃ端的修饰及突变情况。为蛋白质和多肽的完整性、均一性,基因工程产品及合成多肽的质控提供了重要的Ｃ端信息。目前,可在１￣２ｎｍｏｌ水平上测定了３￣５个Ｃ端     

钙激活钾通道在黎芦碱致神经细胞损伤中的作用

目的:观察新生SD大鼠原代培养皮层神经元的钙激活钾通道(Kca)在黎芦碱致神经元损伤模型上的激活、抑制效应.方法:采用细胞贴附和内面向外两种膜片钳单通道记录方法记录新生SD大鼠原代培养皮层神经元的Kca电生理活动.结果:黎芦碱在胞外可激活Kca.在有钙浴液内,细胞贴附式,钳制膜电位 30 mV,加入不同浓度黎芦碱(μmol/L:15、25、50、75),通道开放概率由0.005分别增加为0.014±0.003、0.085±0.010、0.132±0.016、0.059±0.006(P＜0.01),在50μmol/L以内表现出浓度依赖性.无钙浴液内,细胞贴附式膜片上,钳制膜电位 50 mV,随药物浓度(μmol/L)增加为15、40、60、100时,通道开放概率由0.005分别增加为0.014±0.010、0.113±0.006、0.141±0.004、0 295±0.009(P＜0.05).6例内面向外式膜片上,钳制膜电位 40 mV,分别加入黎芦碱25 μmol/L、50μmol/L 3 min后,通道开放概率由0.011±0.008分别增加为0.010±0.010、0.012±0.007(P＞0.05).黎芦碱在胞内Kca开放概率,平均开放/关闭时间,电流幅值均无明显变化.结论:黎芦碱通过影响胞内游离钙水平间接调节Kca,在缺血缺氧早期,胞内游离钙增高激活Kca开放.     

A DNA Vaccine Encoding a Codon-Optimized Human Papillomavirus Type 16 E6 Gene Enhances CTL Response and Anti-tumor Activity

Summary The HPV oncoproteins E6 and E7 are consistently expressed in HPV-associated cancer cells and are responsible for their malignant transformation. Therefore, HPV E6 and E7 are ideal target antigens for developing vaccines and immunotherapeutic strategies against HPV-associated neoplasms. Recently, it has been demonstrated that codon optimization of the HPV-16 E7 gene resulted in highly efficient translation of E7 and increased the immunogenicity of E7-specific DNA vaccines. Since vaccines targeting E6 also represent an important strategy for controlling HPV-associated lesions, we developed a codon-optimized HPV-16 E6 DNA vaccine (pNGVL4a-E6/opt) and characterized the E6-specific CD8⁺ T cell immune responses as well as the protective and therapeutic anti-tumor effects in vaccinated C57BL/6 mice. Our data indicated that transfection of human embryonic kidney cells (293 cells) with pNGVL4a-E6/opt resulted in highly efficient translation of E6. In addition, vaccination with pNGVL4a-E6/opt significantly enhanced E6-specific CD8⁺ T cell immune responses in C57BL/6 mice. Mice vaccinated with pNGVL4a-E6/opt are able to generate potent protective and therapeutic antitumor effects against challenge with E6-expressing tumor cell line, TC-1. Thus, DNA vaccines encoding a codon-optimized HPV-16 E6 may be a promising strategy for improving the potency of prophylactic and therapeutic HPV vaccines with potential clinical implications.     

Determination of the secondary structure of and cellular protein binding to the 3'-untranslated region of the hepatitis C virus RNA genome.

Hepatitis C virus (HCV) contains a positive-stranded RNA genome of approximately 9.5 kb. Despite the overall sequence diversity among individual HCV isolates, the 3'-end 98 nucleotides (nt) of the HCV RNA, which constitute part of the 3'-untranslated region (3'-UTR), are highly conserved. This conserved region may contain the cis-acting signals for RNA replication involving possibly both viral and cellular proteins. We carried out RNase digestion studies, which revealed that this 98-nt region contains three stem-loops but may also assume alternative structures. We further performed UV cross-linking experiments to detect cellular proteins that bound to this region. A 58-kDa cellular protein (p58) was detected. Its binding site was mapped to the stem-loops 2 and 3, which are the most conserved region of the 3'-UTR. Site-directed mutagenesis studies revealed that both stem structures and specific nucleotide sequence within the two loops are important for p58 binding. Mutations that disrupted stem structures abolished protein binding, while the compensatory mutations restored its binding. This region also contains partial sequence similarity to the reported consensus binding sequence for polypyrimidine tract-binding protein (PTB) (a 57-kDa protein). The UV-cross-linked protein could be immunoprecipitated with the anti-PTB antibody, and the recombinant PTB bound to the HCV 3'-UTR with the same binding specificity as p58, establishing that this protein is PTB. However, the reported PTB-binding sequence was not sufficient, but rather the entire stem-loops 2 and 3 were required, for PTB binding; thus, its binding specificity is significantly different from the reported PTB-binding sequence requirement. This protein was detected in both the nuclei and cytoplasm of most mammalian cell lines tested and human primary hepatocytes. PTB may participate in the regulation of HCV RNA synthesis or translation.